We found that LPS causes b-catenin accumulation by glycogen syn-thase kinase 3b GSK3b inactivation, and that b-catenin accumulation increases Nox1 expression.. LPS induced Nox1 mRNA expr
Trang 1involved in toll-like receptor 4-mediated NADPH oxidase 1 expression in macrophages
Jin-Sik Kim1*, Seungeun Yeo1*, Dong-Gu Shin2, Yoe-Sik Bae3, Jae-Jin Lee4, Byung-Rho Chin4, Chu-Hee Lee1and Suk-Hwan Baek1
1 Department of Biochemistry & Molecular Biology, Yeungnam University, Daegu, Korea
2 Department of Internal Medicine, Yeungnam University, Daegu, Korea
3 Department of Biochemistry, Dong-A University, Busan, Korea
4 Department of Dentistry, Yeungnam University, Daegu, Korea
Introduction
NADPH oxidase (Nox) is an essential enzyme in
phagocytes and generates reactive oxygen species
(ROS) for host defense [1] Extensive evidence has now
shown that Nox is also one of the major sources of
ROS production in various nonphagocytic cells,
including smooth muscle cells and hepatocytes [2,3]
Members of the Nox family are transmembrane
pro-teins that catalyze the NADPH-dependent one-electron
reduction of oxygen to form superoxide [4] To date,
seven members of this family have been described: Nox1–5 and dual oxidase (Duox)-1 and -2 Among these, the most studied is Nox2
Nox1, the first-recognized homologue of Nox2 [5], conserves the structural domain common to the cata-lytic core of Nox2 Nox1-derived ROS were initially reported to have tumorigenic and angiogenic functions [6,7] However, further studies suggest that Nox1 regu-lates inflammation [8], atherosclerosis [9] and vascular
Keywords
glycogen synthase kinase 3b; NADPH
oxidase 1; reactive oxygen species; toll-like
receptor 4; b-catenin
Correspondence
Suk-Hwan Baek, Department of
Biochemistry & Molecular Biology, College
of Medicine, Yeungnam University, 317-1
Daemyung-5 Dong, Daegu 705-717, Korea
Fax: +82 53 623 8032
Tel: +82 53 620 4523
E-mail: sbaek@med.yu.ac.kr
*These authors contributed equally to this
work
(Received 25 February 2010, revised 23
April 2010, accepted 27 April 2010)
doi:10.1111/j.1742-4658.2010.07700.x
Macrophage activation contributes to the pathogenesis of atherosclerosis
In the vascular system, the major source of reactive oxygen species is the NADPH oxidase (Nox) family Nox1 is induced by lipopolysaccharide (LPS) in macrophages, but the expression mechanism is not fully under-stood We found that LPS causes b-catenin accumulation by glycogen syn-thase kinase 3b (GSK3b) inactivation, and that b-catenin accumulation increases Nox1 expression LPS induced Nox1 mRNA expression and reac-tive oxygen species generation in Raw264.7 cells Using bone marrow-derived macrophages from toll-like receptor 4 mutant mice, we also tested whether LPS-induced Nox1 expression is toll-like receptor 4 dependent LPS caused GSK3b phosphorylation, induced b-catenin accumulation and increased nuclear translocation The GSK3b inhibitor LiCl potentiated LPS-induced Nox1 expression in accordance with b-catenin accumulation and nuclear translocation Conversely, ectopic expression of a constitutively active GSK3b mutant severely attenuated Nox1 expression These findings identify a novel regulatory pathway controlling Nox1 expression by LPS-stimulated macrophages
Abbreviations
BMDMs, bone marrow-derived monocytes; DAPI, 4¢,6-diamidino-2-phenylindole; GSK3b, glycogen synthase kinase 3b; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; Nox, NADPH oxidase; ROS, reactive oxygen species; TLR, toll-like receptor.
Trang 2tone [10] It has been shown to regulate smooth muscle
cell growth, both hypertrophy and hyperplasia, and
migration [11] In addition, Nox1 may be important in
regulating blood pressure [12] Furthermore, Nox1
shows a relationship with toll-like receptor (TLR) in
controlling innate immunity Kawahara et al [13] have
shown that lipopolysaccharide (LPS) from pathogenic
Helicobacter pyloristrains may potently stimulate ROS
production, mediated by Nox1, through a
TLR4-dependent pathway Lee et al [14] also reported that
the TLR9 agonist CpG ODN causes ROS production
via Nox1 gene expression in macrophages and
contrib-utes to foam cell formation These studies suggest a
crucial role for Nox1 in TLR-mediated signaling
path-ways and its importance in innate immunity and
inflammatory responses
The regulation of Nox1 seems to differ significantly
from that of Nox2, and the Nox1 regulatory
mecha-nism is still poorly defined Full Nox1 activity is
known to need regulatory cofactors, including NoxO1,
NoxA1 and Rac1 [5] The most well-studied activation
of Nox1 is that of angiotensin II mediating
phospholi-pase C and protein kinase C in vascular smooth
mus-cle cell However, regulation of Nox1 activity by
mRNA induction is also important Nox1 mRNA is
most highly expressed in colon epithelia [15], but is
also expressed at lower levels in macrophages [16]
Known Nox1-inducing factors are TLR agonists such
as angiotensin II, interferon-c and platelet-derived
growth factor [17–20] However, research on the
essen-tial proteins involved in Nox1 gene inducement in
macrophages is not clear There have been some
reports of the Nox1 expression mechanism in
macro-phages Reports show that IRAK-1 increases Nox1
expression to produce ROS [21], and the increase in
Nox1-mediated ROS production is involved in
macro-phage differentiation into receptor activator of NF-j-B
ligand (RANKL)-induced osteoclasts [22] Also, we
have previously reported that activation of c-Jun
NH2-terminal kinase (JNK) and cytosolic
phospho-lipase A2 (cPLA2) by CpG ODN is essential in Nox1
mRNA expression and ROS generation in
macrophag-es [23] Recently, we reported that calcium-independent
phospholipase A2b-mediated signaling regulates Nox1
expression in macrophages, and that the ROS
pro-duced are part of an important process controlling
foam cell formation [24]
Glycogen synthase kinase 3 (GSK3)b and the
b-cate-nin pathway are crucial regulators in the balance
between pro- and anti-inflammatory cytokine
produc-tion [25] Recently, this pathway was shown to have
an essential role in inflammation and immune cells
[25] In particular, many groups have shown that
GSK3b, through TLR signaling, is necessary in inflam-mation For example, it has been reported that GSK3b regulates TLR-mediated cytokine production and inac-tivation of GSK3b by LPS has a negative effect on production of the pro-inflammatory cytokine inter-feron-b [26] b-Catenin is one of the most important downstream molecules of the GSK3b pathway [27] When b-catenin is released into the cytosol and is not degraded by the proteosome, it may be translocated to the nucleus and form a complex with T-cell factor The b-catenin⁄ T-cell factor complex acts as a tran-scriptional activator of many genes [28]
In this study, we show that GSK3b plays a funda-mental role in regulating Nox1 gene expression LPS causes phosphorylation of GSK3b and accumulation
of b-catenin Inhibition of GSK3b activity potently augmented expression of the Nox1 gene in LPS-stimu-lated macrophages, whereas ectopic expression of a constitutively active GSK3b mutant reduced Nox1 gene expression Taken together, these findings suggest that GSK3b and the b-catenin pathway are critical reg-ulators controlling Nox1 gene expression
Results
Stimulation of macrophages with LPS induces the expression of Nox1 via TLR4
We studied Nox1 expression and ROS formation by TLR4 agonist LPS in Raw264.7 cells As shown previ-ously [24], LPS increased Nox1 mRNA expression in a time-dependent manner (Fig 1A) and induced ROS formation (Fig 1B) The TLR4 dependence of LPS-induced Nox1 expression was confirmed using TLR4 mutant mice Monocytes separated from the bone mar-row of TLR4 wild-type (C3H⁄ HeN) and TLR4 mutant (C3H⁄ HeJ) mice were differentiated into macrophages
by adding macrophage colony-stimulating factor (M-CSF) We stimulated each bone marrow-derived monocyte (BMDM), isolated from two types of mice, with LPS, and compared the Nox1 mRNA expression using RT-PCR The effects of LPS were strong in the BMDM of C3H⁄ HeN, but relatively weak in the BMDM of C3H⁄ HeJ (Fig 1C) The data suggest that TLR4 is involved in LPS-induced Nox1 expression
LPS phosphorylates GSK3b via TLR4 in macrophages
LPS stimulation of innate immune cells has been shown
to promote GSK3b inactivation via phosphorylation of serine 9 (S9) [26] Therefore, we tested changes in GSK3b phosphorylation, using LPS stimulation, in
Trang 3Raw264.7 cells LPS stimulation induced GSK3b
phos-phorylation (Fig 2A,B) To assess whether TLR4 is
required for LPS to induce GSK3b phosphorylation,
TLR4 wild-type and mutant macrophages were
com-pared for their abilities to phosphorylate GSK3b upon
LPS stimulation GSK3b phosphorylation increased in
the wild-type, but was expressed relatively weakly in the
mutant type (Fig 2C)
LPS induces b-catenin accumulation and nuclear
translocation
Because LPS induced phosphorylation of GSK3b, we
investigated whether b-catenin accumulation was
affected by LPS stimulation LPS induced b-catenin
accumulation of macrophages and showed maximum effects at 45 min with dose dependency (Fig 3A,B)
To confirm the subcellular localization of b-catenin,
we fractionated cells by cytosol and nuclear fraction after LPS stimulation The time course of subcellular localization for b-catenin showed a progressive increase in the cytosolic fraction and a concomitant significant increase in the nuclear fraction (Fig 3C), suggesting that b-catenin was translocated into the nucleus These results were also supported by fluores-cent microscopy In untreated cells, b-catenin was mainly localized in the cytosol at a low level After
45 min of treatment with LPS, the distribution of b-catenin in the subcellular compartments was altered,
as shown by the nuclear translocation (Fig 3D) This shows that LPS is able to induce b-catenin accumula-tion and subsequent translocaaccumula-tion into the nucleus
C
Nox1
-actin
0 0.5 1 2 4 6 0 0.5 1 2 4 6 LPS (h)
C3H/HeN (TLR4 WT) C3H/HeJ (TLR4 Mut)
A
Nox1
β
ββ
-actin
0 1 2 3 4 6 LPS (h)
B
0
0.4
0.8
1.2
0 60 120 180 240 300
LPS Time (min)
Con LPS
Fig 1 Induction of Nox1 mRNA and production of ROS by LPS in
macrophages (A) Raw264.7 cells were stimulated with LPS
(100 ngÆmL)1) for the indicated times Nox1 mRNA expression
was determined by RT-PCR and was normalized to b-actin (B)
Raw264.7 cells were cultured in 96-well plates in a CO2incubator
for 1 h The cells were changed to NaCl ⁄ P i containing lucigenin
(100 l M ) and NADPH (200 l M ) and treated with LPS (100 ngÆmL)1)
for 300 min Chemiluminescence was measured in relative light
units (RLU) every 10 min over a period of 300 min (C) Primary
BMDMs were isolated from C3H ⁄ HeN (TLR4 wild-type)
or C3H ⁄ HeJ (TLR4 mutant) mice BMDMs were differentiated for
5–7 days in media containing M-CSF and stimulated with LPS
(100 ngÆmL)1) for the indicated times Nox1 mRNA expression was
determined by RT-PCR and was normalized to b-actin The data are
representative of three independent experiments.
0 10 50 100 500 LPS (ng·mL –1 )
pGSK3ββ
GSK3β
B
A
pGSK3β
GSK3β
0 15 30 45 60 LPS (min)
1 1.6 1.9 1.4 0.7 (fold)
C
pGSK3β
GSK3β
HeN (TLR4 WT) HeJ (TLR4 mut)
1 1.8 1.3 0.7 0.5 0.9 0.7 0.6 (fold)
Fig 2 Inactivation of GSK3b by LPS in macrophages (A,B) Raw264.7 cells were incubated with LPS (100 ngÆmL)1) for different times or doses To assess phospho-GSK3b (S9), total cell lysates were resolved on SDS ⁄ PAGE, immunoblotted with anti-(phospho-GSK3b) or anti-GSK3b sera, and developed using enhanced chemiluminescence (C) Wild-type (C3H ⁄ HeN) and TLR4 mutant (C3H ⁄ HeJ) BMDMs were stimulated with LPS (100 ngÆmL)1) for the indicated times Phosphorylated GSK3b expression was determined by western blot using anti-(phospho-GSK3b) serum and was normalized to GSK3b expression The data are represen-tative of three independent experiments.
Trang 4GSK3b negatively controls Nox1 expression by
LPS-stimulated macrophages
Because LPS induced GSK3b phosphorylation and
b-catenin accumulation, we investigated whether this
pathway played a role in Nox1 gene expression LiCl
is a well-known pharmacologic GSK3b inhibitor
GSK3b-inactivated macrophages stimulated with LPS
produced significantly more b-catenin protein which was also translocated more into the nucleus, than cells stimulated with LPS alone (Fig 4A,B) Also, Nox1 expression, using LPS plus LiCl, showed a greater increase than LPS alone (Fig 4C) The function of GSK3b was confirmed in Nox1 expression using a constitutively active GSK3b (S9A) mutant Green fluorescent protein-tagged GSK3b (S9A) mutant was successfully overexpressed and the accumulation of b-catenin and translocation to the nucleus by LPS decreased more than in cells transfected with only the vector (Fig 4D–F) Taken together, these results dem-onstrate that GSK3b plays a fundamental role in con-trolling the expression of Nox1 by TLR4-stimulated macrophages
Discussion ROS are known to act as a signaling molecule in vari-ous physiological processes because of their regulated production by ligands, the existence of catabolic metabolism to terminate their signaling and their redox-dependent reversible modification of target pro-teins [29] ROS are also considered important in mac-rophage activation, because this process is significantly related to the pathogenesis of inflammatory diseases such as atherosclerosis or metabolic syndrome
The ROS production system of macrophages is vari-able and complicated, and Nox and its function have become current issues Seven types of Nox have been found The most studied is Nox2; however, other types, especially Nox1 and Nox4, are also active areas
of research ROS produced by Nox has a general downstream physiological role ROS produced by Nox2 is required in the respiratory burst that occurs in phagocytes [30] It has been suggested that other types
of Nox are needed in host defense For example, Nox1
is important in the colon and Duox-1 and -2 are important in the lung [31] Nox1 also contributes sig-nificantly to gastrointestinal inflammation, hyperten-sion and restenosis after angioplasty development [8,18] ROS production from Nox1 activity is primarily controlled by p22phox and the Nox1 regulators NoxA1 and NoxO1 [5], but an increase in Nox1 gene expression is also essential Angiotensin II and plate-let-derived growth factor lead to increased Nox1 mRNA levels and contribute to vascular pathology [18,20] The TLR agonists LPS, flagellin and CpG ODN have been shown to be Nox1 gene-inducing fac-tors, leading to the verification of Nox1 function in immune responses and the development of atheroscle-rosis [17] Therefore, the regulation of Nox1 mRNA expression may be a potential therapeutic target for
C
β-catenin (short) β-catenin (long) β-tubulin
0 15 30 45 60 0 15 30 45 60 LPS (min)
Cytosol Nucleus
D DAPI FITC Merge
Con
LPS
B
0 10 50 100 500 LPS (ng·mL –1 )
ββ-catenin β-actin
A
0 15 30 45 60 LPS (min)
β-catenin β-actin
Fig 3 Changes in the subcellular localization of b-catenin induced
by LPS in macrophages (A,B) Raw264.7 cells were incubated with
LPS (100 ngÆmL)1) for different times or doses To assess b-catenin,
total cell lysates were resolved on SDS ⁄ PAGE, immunoblotted with
an anti-(b-catenin) serum and developed using enhanced
chemilumi-nescence (C) Cells were treated with LPS (100 ngÆmL)1) for the
indicated times and fractionated into cytosolic and nuclear extracts.
Western blot on both extracts was determined by using
anti-(b-catenin) serum b-Tubulin served as loading control and nuclear
marker (D) Macrophages were treated with LPS (100 ngÆmL)1) for
40 min and stained with anti-b-catenin and DAPI at room
tempera-ture for 10 min The cells were washed three times with NaCl ⁄ P i
The images were acquired and analyzed using fluorescent
micros-copy The data is representative of five independent experiments.
Trang 5these diseases However, more research into the
mecha-nism controlling Nox1 mRNA expression is needed
We previously reported that various types of TLR
agonists increase ROS production by inducing Nox1
mRNA expression, and the increased ROS convert
macrophages to foam cells by low-density lipoprotein
oxidation [14] The aim of this study was to find the
signaling transduction molecule contributing to Nox1
mRNA expression by LPS Our results showed that
GSK3b is a very important factor in b-catenin
signal-ing In other words, LPS inactivates GSK3b through
phosphorylation, and the inactivated GSK3b inhibits
the degradation of b-catenin, promoting translocation
into the nucleus It is hypothesized that the
translocat-ed b-catenin forms a complex with a specific
transcrip-tion factor, and thereby leads to an increase in Nox1
mRNA expression Furthermore, experimental results,
using GSK3b inhibitor and constitutively active
GSK3b, have shown the importance of GSK3b in
Nox1 mRNA regulation
Signaling molecules, such as protein kinase C-d (PKC-d) and calcium-independent phospholipase A2 (iPLA2b), are known to regulate Nox1 mRNA [24,32]
We previously reported that Akt also regulates Nox1 mRNA expression [24] Therefore, we investigated the correlation between Akt and GSk3b The Akt inhibitor LY294002 inhibited GSK3b phosphorylation by LPS and also decreased Nox1 mRNA expression (Fig S1) These results suggest that LPS induces Akt phosphory-lation and the activated Akt inactivates GSK3b, thereby regulating Nox1 gene expression However, various signaling proteins that control Akt exist and it
is believed that more proteins may participate in the process, showing the need for further research
Consequently, LPS induces Nox1 expression via TLR4 Phosphorylation of GSK3b by LPS inactivates GSK3b and increases translocation of b-catenin to the nucleus by inhibiting degradation We suggest that translocated b-catenin will activate specific transcrip-tion factors and eventually increase Nox1 mRNA
C
Nox1
-actin
B
Con
Merge FITC
DAPI
LPS
LiCl
LPS LiCl
A
-catenin -actin
D
-catenin
EGFP-GSK3
GSK3
Vec GSK3
S9A
F
-actin Nox-1
S9A
DAPI TRITC Merge
-LPS
-LPS Vector
GSK3 S9A
E
Fig 4 The effect of GSK3b on LPS-induced b-catenin and Nox1 expression (A–C) Raw264.7 cells were stimulated with LPS (100 ngÆmL)1) in the presence or absence of the GSK3b inhibitor, LiCl (5 l M ) (A) The cell lysates were analyzed for b-catenin using western blotting (B) Cells were fixed and stained with anti-(b-catenin) serum and DAPI and observed using fluorescent microscopy (C) Nox1 mRNA was analyzed by RT-PCR (D–F) Raw264.7 cells were transfected with
a pEGFP-C1 vector expressing a constitu-tively active form (GSK3b S9A) or vector alone Gene-transfected cells were stimu-lated with or without LPS (100 ngÆmL)1) (D) Cell lysates were analyzed for GSK3b, b-catenin by western blotting (E) Cells were fixed and stained with anti-(b-catenin) serum and DAPI and observed with fluorescent microscopy (F) Nox1 mRNA was analyzed
by RT-PCR The data are representative of five independent experiments.
Trang 6expression Our results showing the possibility of the
Nox1 gene being regulated by GSK3b⁄ b-catenin are
ori-ginal and lead to the possibility that GSK3b⁄ b-catenin
may contribute to the development of atherosclerosis
Materials and methods
Reagents
Cell culture reagents, including fetal bovine serum, were
obtained from Life Technologies (Grand Island, NY,
USA) GSK3b, b-catenin and b-actin antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) and
phospho-GSK3b, Akt, p-Akt antibodies were from Cell
Signaling Technology (Danvers, MA, USA) Escherichia coli
LPS (0111:B4, Cat No: L3024, purified by ion-exchanged
chromatography and containing > 1% protein and RNA),
NADPH and lucigenin were from Sigma-Aldrich (St
Louis, MO, USA), the RT-PCR kit was from Takara Bio
mutant (C3H⁄ HeJ) mice were purchased from Central Lab
Animal Inc (Seoul, Korea) 4¢,6-diamidino-2-phenylindole
(DAPI) and Alexa fluor 488 goat anti-(rabbit IgG) were
(TRITC)-conjugated AffiniPure donkey anti-(rabbit IgG)
was from Jackson ImmunoResearch Laboratories Inc
(West Grove, PA, USA)
Plasmids and transfection
In order to make a GSK3b (S9A) plasmid construction,
cDNA from Raw264.7 cells was amplified by PCR with
mutation primer-1 (forward: 5¢-ACTCCACCCTTTTTCTC
CTC-3¢, reverse: 5¢-GCTCTCCGCAAAGGCGGTGGT-3¢)
and mutation primer-2 (forward: 5¢-CGACCGAGAACCA
CCGCCTTTGC-3¢, reverse: 5¢-CGCGTCGACCTCCTGG
GGGCTGTTCAG-3¢) Two PCR products were mixed and
amplified by cloning primer (forward: 5¢-CGCAGATCTA
TGTCGGGGCGACCGAGA-3¢, reverse: 5¢-CGCGTCGA
CCTCCTGGGGGCTGTTCAG-3¢) to obtain insert cDNA
Insert cDNA was ligated with pEGFP-C1 vector (Invitrogen)
and the ligated vector was transformed into DH5a cells
Nucleotide sequencing was performed after plasmid
prepara-tion Cells were transfected with pEGFP-C1–GSK3b (S9A)
plasmid using Lipofectamine LTX reagent (Invitrogen)
according to the manufacturer’s protocol and then incubated
for 24 h before LPS stimulation
Cell culture and mouse BMDM preparation
The Raw264.7 macrophage cell line was obtained from the
American Type Culture Collection (Manassas, VA, USA)
and cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and 1%
for 5–7 days in media containing M-CSF The culture med-ium consisted of Dulbecco’s modified Eagle’s medmed-ium sup-plemented with 10% L929 cell-conditioned medium (as a source of M-CSF) This study was conducted in accordance with the guidelines for the care and use of laboratory ani-mals provided by Yeungnam University and all experimen-tal protocols were approved by the Ethics Committee of Yeungnam University, South Korea
Lucigenin assay
NADPH-dependent ROS generation was measured by monitoring lucigenin-derived chemiluminescence at room temperature using the Lmax II luminometer (Molecular Devices, Sunnyvale, CA, USA) Briefly, cells were cultured
in 96-well plates, pretreated with LiCl for 1 h, subsequently
(100 lm) and NADPH (200 lm) LPS was added exoge-nously to the suspended cells Chemiluminescence was mea-sured in relative light units every 10 min over a period of 200–300 min
Fluorescent microscopy assay
Cells were plated in 24-well plates containing embedded glass cover slips and pretreated with LiCl for 1 h before
fixed and stained with anti-b-catenin serum and DAPI and observed using fluorescent microscopy Fluorescent micros-copy images were acquired and analyzed using an Olympus BX51 fluorescent microscope and DP Manager Software (Olympus, Japan)
Cytosolic and nuclear fractionation
Cells were plated in a 100 mm diameter dish and pretreated
for the indicated times Cytosolic and nuclear fractionation was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according
to manufacturer’s protocol
RT-PCR
Total RNA was extracted from cells using Trizol reagent (Invitrogen) One microgram of total RNA was used as a template to make first-strand cDNA by oligo(dT) priming using a commercial reverse transcriptase system (Promega, Madison, WI, USA) The synthetic gene-specific primer sets used for PCR were Nox1 forward primer, 5¢-AAGTGGCT GTACTGGTTGG-3¢, and reverse primer, 5¢-GTGAGGA
Trang 7AGAGTCGGTAGTT-3¢, which amplified 238 bp of mouse
Nox1 cDNA, and b-actin forward primer, 5¢-TCCTTCGT
TGCCGGTCCACA-3¢, and reverse primer, 5¢-CGTCTCC
GGAGTCCATCACA-3¢, which amplified 509 bp of mouse
normalized against b-actin
Western blot analysis
Macrophages were cultured in six-well plates and treated
with LPS in the presence or absence of an inhibitor Cell
pH 8.0, 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40,
1 mm phenylmethanesulfonyl fluoride, and protease
inhibi-tor cocktail) Proteins were separated by 8% reducing
mem-branes in 20% methanol, 25 mm Tris and 192 mm glycine
Membranes were then blocked with 5% non-fat dry milk
and incubated with primary antibody overnight The
mem-branes were washed, incubated for 1 h with a secondary
antibody conjugated to horseradish peroxidase, rewashed
and developed using an enhanced chemiluminescence
sys-tem (GE Healthcare, Chalfont St Giles, UK)
Acknowledgement
This work was supported by the Korean Science and
Engineering Foundation via the Aging-associated
Vas-cular Disease Research Center at Yeungnam University
(R13-2005-005-02001-0)
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Supporting information The following supplementary material is available: Fig S1 The effect of Akt on LPS-induced GSK3b inactivation and Nox1 expression
This supplementary material can be found in the online version of this article
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